Abstract
Immobilized enzymes have a very large region that is not in contact with the support surface and this region could be the target of new stabilization strategies. The chemical amination of these regions plus further cross-linking with aldehyde-dextran polymers is proposed here as a strategy to increase the stability of immobilized enzymes. Aldehyde-dextran is not able to react with single amino groups but it reacts very rapidly with polyaminated surfaces. Three lipases—from Thermomyces lanuginosus (TLL), Rhizomucor miehiei (RML), and Candida antarctica B (CALB)—were immobilized using interfacial adsorption on the hydrophobic octyl-Sepharose support, chemically aminated, and cross-linked. Catalytic activities remained higher than 70% with regard to unmodified conjugates. The increase in the amination degree of the lipases together with the increase in the density of aldehyde groups in the dextran-aldehyde polymer promoted a higher number of cross-links. The sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) analysis of those conjugates demonstrates the major role of the intramolecular cross-linking on the stabilization of the enzymes. The highest stabilization was achieved by the modified RML immobilized on octyl-Sepharose, which was 250-fold more stable than the unmodified conjugate. The TLL and the CALB were 40-fold and 4-fold more stable than the unmodified conjugate.
Highlights
Enzymes are nature’s catalysts for carrying out very complex chemical reactions
The aldehyde-dextran-Toyopearl (ADT) support was prepared to demonstrate that the aldehydes of an oxidized dextran polymer present the same chemical reactivity as the aldehyde groups of a glyoxyl-agarose support
In the presence of dithiothreitol (DTT), the one-point amino-aldehyde attachments become irreversible. This stabilizing effect of DTT on single Schiff’s bases has been previously reported [34]. This means that the amino terminus of the penicillin G acylase (PGA) is exposed to the medium but that it is unable to react by one-point attachment with aldehyde groups in the absence of stabilizers
Summary
Enzymes are nature’s catalysts for carrying out very complex chemical reactions They have excellent catalytic properties such as high activity, selectivity, and specificity. Each aldehyde-dextran molecule could form several stabilizing two-point stable attachments with amino groups on enzyme surfaces. The main property of these aldehyde groups is their high reactivity towards N-nucleophiles as amino groups, yielding very unstable one-point attachments but very stable two-point attachments when the attachment simultaneously involves two vicinal groups [20]. The model enzyme chosen for this experiment was penicillin G acylase (PGA) from E. coli These polymers were used to stabilize three highly aminated immobilized lipases from Themomyces lanuginosus (TLL) [22], Rhizomucor miehiei (RML) [23], and Candida antarctica B (CALB) [24]. We took advantage of this feature to protect the lipase’s active center from possible undesirable modifications with aldehyde-dextran
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